The highly conserved retromer complex regulates the recycling of cell-surface membrane proteins. Defects in retromer function have been linked to Alzheimer?s disease and Parkinson?s disease (PD). Mutations in retromer component Vps35 underly some cases of late-onset PD. However, because vesicular trafficking defects in the endolysosomal pathways occur in cells expressing alpha-synuclein (?-syn) and that have no Vps35 mutations ?-syn itself perturbs retromer function. Our preliminary results show that ?-syn disrupts retromer function under certain conditions and hence affects vesicular trafficking. The long-term goal of this proposed research is to increase our understanding of how ?-syn modulates retromer-mediated endocytic recycling. ?-syn has been implicated in both early-onset and sporadic PD. Our central hypothesis, based on our preliminary results, is that ?-syn disrupts Snx3-retromer-mediated recycling of membrane-bound proteins by inhibiting the binding of retromer component sorting nexin 3 (Snx3) and Vps17 to liposomes containing phosphatidylinositol 3?-phosphate (PI3P). Snx3 is a cargo-specific adapter protein that contains a PI3P-binding module called a phox homology domain (PX). We have obtained data using a yeast PD model that ?-syn blocks the binding of Snx3 to early endosomes, resulting in the failure of Snx3 to engage its cargo proteins Fet3/Ftr1, and instead of recycling endocytosed Fet3/Ftr1 back to the plasma membrane the complexes transit to the vacuole for degradation. Our hypothesis is that ?-syn inhibits the binding of the Snx3 PX domain to PI3P embedded in early endosomes. The specific aims are to (1) determine whether ?-syn prevents Snx3 from interacting with its other bona fide substrates, that is, the Golgi resident proteins Kex2 and Ste13. This aim will be accomplished by monitoring changes in the localization of Kex-GFP in yeast cells ?- syn by both fluorescence microscopy and transmission electron microscopy. (2) We will determine whether elevated expression of ?-syn inhibits the binding of two yeast proteins that possess a different PI3P binding module called a ?FYVE? domain; these two FYVE-containing proteins are Vac1 and Vps27, which orchestrate vesicular trafficking from endosomes to the vacuole/lysosome. The hypothesis is that at elevated concentrations ?-syn not only disrupts PX domain proteins binding to PI3P, it also disrupts FYVE domain containing proteins to PI3P in early/late endosomes. Fluorescence and electron microscopies will enable us to follow the fate of these proteins. (3) We will probe the mechanism by which ?-syn inhibits Snx3 association with vesicles using an in vitro liposome?protein binding assay, particularly evaluating the importance of interactions between phosphatidylserine and the PX and FYVE domains vis--vis vesicle binding. Lastly, we will test whether the flexible, C-terminal tail of ?-syn contributes to ?-syn?s ability to block the association of PX (and FYVE) domains with PI3P. The results will lay the foundation for advances in disease diagnosis, treatment, and prevention.